Detection of Paternally Inherited Fetal Point Mutations for β-Thalassemia Using Size-Fractionated Cell-Free DNA in Maternal Plasma

Abstract

Context Currently, fetal point mutations cannot be reliably analyzed from circulatory fetal DNA in maternal plasma, due to the predominance of maternal DNA sequences. However, analysis of circulatory fetal DNA sequences in maternal plasma have been shown to selectively enrich for fetal DNA molecules on the basis of a smaller molecular size than maternal DNA.Objective To examine the prenatal analysis of 4 common ␤-thalassemia point mutations: IVSI-1, IVSI-6, IVSI-110, and codon 39.Design, Setting, and Patients A total of 32 maternal blood samples were collected at 10 to 12 weeks of gestation (mean, 10.7 weeks) between February 15, 2003, and February 25, 2004, in Bari, Italy, from women with risk for ␤-thalassemia in their newborns immediately prior to chorionic villous sampling. Samples in which the father and mother did not carry the same mutation were examined. Circulatory DNA was size-fractionated by gel electrophoresis and polymerase chain reaction (PCR) amplified with a peptide-nucleic-acid clamp, which suppresses amplification of the normal maternal allele. Presence of the paternal mutant allele was detected by allele-specific real-time PCR.Main Outcome Measure Detection of paternally inherited ␤-globin gene point mutations.Results Presence or absence of the paternal mutant allele was correctly determined in 6 (86%) of 7 cases with the IVSI-1 mutation, 4 (100%) of 4 with the IVSI-6 mutation, 5 (100%) of 5 with the IVSI-110 mutation, and 13 (81%) of 16 with the codon 39 mutation. One false-positive test result was scored for the IVSI-1 mutation. Two cases with the codon 39 mutation were classified as uncertain and 1 case was excluded due to lack of a diagnostic test result at the time of analysis. These results yielded an overall sensitivity of 100% and specificity of 93.8%, with classified cases removed. ConclusionOur recently described technique of the size-fractionation of circulatory DNA in maternal plasma may be potentially useful for the noninvasive prenatal determination of fetal point mutations.

Next generation sequencing of SNPs for non-invasive prenatal diagnosis: challenges and feasibility as illustrated by an application to β-thalassaemia

“…[17][18][19][20] In view of this limitation, we previously showed that the detection of the paternally inherited SNPs is feasible for the NIPD of b-thalassaemia. 22,23 SNPs can be used regardless of the mutation of the carrier couples, they provide positive detection of the paternal allele, normal or mutant, the result can be confirmed with more than one SNP and, importantly, the more SNPs used the less diagnostic risk.…”

“…Allele-specific real-time PCR is one of the first approaches that have been used to exclude paternal mutations in the maternal circulation. 17 Preferential detection of fetal alleles was achieved through initial enrichment of fetal DNA, 8,18 while others enhanced the production of the mutated fetal allele by employing either peptide nucleic acid probes 19 or COLD PCR. 20 In the specific case of b-thalassaemia, MALDI-TOF mass spectrometry has been also investigated.…”

“…Hence, despite the advances in technology, NIPD of b-thalassaemia has yet to reach clinical practice. 8,18,19,22,23,25 Our approach is based on the detection of the paternally inherited alleles as maternal alleles cannot be differentiated from fetal ones. Therefore, NIPD is possible only in those cases where the fetus inherits the normal allele of the father.…”

A Minimal Set of SNPs for the Noninvasive Prenatal Diagnosis of β‐Thalassaemia

“…Allele-specific real-time polymerase chain reaction (PCR) is one of the first approaches that have been used to exclude paternal mutations in the maternal circulation (Chiu et al, 2002). Preferential detection of fetal alleles was achieved through initial enrichment of fetal DNA (Li et al, 2005;Li et al, 2009), whereas others enhanced the production of the mutated fetal allele by employing either peptide nucleic acid (PNA) probes (Galbiati et al, 2008) or COLD PCR (Galbiati et al, 2011). In the specific case of β-thalassaemia, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has also been investigated (Li et al, 2009).…”

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“…Although recent reports have indicated that it may be possible to noninvasively detect fetal aneuploidies such as Down syndrome (trisomy 21) (Lo et al, 2007) or Mendelian single gene disorders, such as the hemoglobinopathies (Ding et al, 2004;Li et al, 2005;Galbiati et al, 2008), no test has yet been implemented in a clinical setting.…”